Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

contamination in transformed bacteria


  • Please log in to reply
8 replies to this topic

#1 cotchy

cotchy

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 87 posts
1
Neutral

Posted 03 March 2009 - 01:55 AM

hi all,

Just a quick question.

Has anybody ever encountered the problem of black filamentous fungi (or at least what looks like fungi) in culture medium when growing competent bacterial cells containing plasmid of interest?

I followed normal protocols for culturing bacteria on antibiotic resistant plates and the bacteria containing the plasmid was supplied by addgene and this is the first time it has been used and all techniques were aseptically carried out.

But after 6 hours of growth in the antibiotic LB broth my bacteria had grown p[retty well (measured as turbidity in broth) but also some black stringy what looked like fungi had grown (but not very much) and was present in all 5 separate duran bottles containing my bacteria.

Does anyone know what this is and will it affect my plasmid as i awill be extracting using a qiagen kit later and pressume the lysis and DNA removal will also removed the contamination DNA?

Can anyone offer some advice?

#2 GeorgeWolff

GeorgeWolff

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 294 posts
0
Neutral

Posted 03 March 2009 - 02:38 AM

There certainly are black fungi - also called dematiceous - such as Alternaraia and Cladosporium and they're very common. These typically wouldn't grow much after just 6 hours. Plate out some of hyphal material and see if it grows.

If indeed these are fungi I suggest you work on your aseptic technique and work in the hood. You might also leave some agar plates open on the relevant benchtop for a brief period to get a feel for air quality at that point.

#3 gebirgsziege

gebirgsziege

    I like fungi

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 392 posts
25
Excellent

Posted 03 March 2009 - 03:04 AM

There is quite a number of black fungi....but as George mentioned they won't grow much within 6 hours.

I am not sure if they would effect your plasmid preperation....this surly depends on your lysis method and the way of your plasmid purification and what gene/protein etc. you are interested in...and if there is a risk of interference or wrong results becaus of fungi.

In contrast to bacteria fungi have a cell wall which can be very stable so it might be that you do not even open up enough fungal cells during lysis to get near a problem with your plasmid.
If you want to use your bacterial cultures: maybe you can try some kind of size exclusion filtration with a filter size big enough for bacteria but too small for fungi to pass? So you could get rid of most of the hyphae and reduce the risk of contamination with fungal DNA although there will always be a rest of fungi in the filtrate.

Otherwise: streak out your culture, and like George suggested re-grow it under aseptic conditions and be sure your medium is not contaminated.
A man cannot be too careful in the choice of his enemies. (Oscar Wilde)

#4 GeorgeWolff

GeorgeWolff

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 294 posts
0
Neutral

Posted 03 March 2009 - 03:30 AM

Agree with my colleague gebirg. and emphasize the point that trying to recover this experiment is very sloppy science.

Determine if it is a fungus or some inanimate material so you can avoid it next time. If it is a fungus, it had to be the product of the material used (check the culture purity) or your technique - don't figure spontaneous generation has happened again.

In any case, scrap this experiment and start over.

Edited by GeorgeWolff, 03 March 2009 - 03:31 AM.


#5 cotchy

cotchy

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 87 posts
1
Neutral

Posted 03 March 2009 - 04:19 AM

Ok guys, thanks very much for the advice,

However, I have already started with the purification procedure so i will keep it going this once too see if there is contamination.

There are some steps of the (Qiagen plasmid midi kit) purification that i think will favour me to get a positive outcome such as,

Firstly i centrifuged the whole suspension at 2500 rpm which pellted (most of not all of) the fungi but not the bacteria and I removed the supernatant and placed the pellet to waste

Also during the purification steps there are high speed centrifugations (remove the fungi, either whole or partially lysed, as you mentioned lysis buffer may not work and also cellular proteins etc),

DNAses (remove bacterial and fungal DNA),

Charged column filtrations (allow only very small molecules through and are charged to bind only RNA which is eluted after)

Running a gel electrophoresis of my eluate should tell me if any fungal RNA remains if so i will scrap the lot.

So as i said i will see how this one goes but also improve my technique for the next attempts

thanks again.

Cotchy.

Edited by cotchy, 03 March 2009 - 04:20 AM.


#6 aimikins

aimikins

    Sunflower

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 117 posts
2
Neutral

Posted 03 March 2009 - 11:03 AM

your best bet will always be to step back and make sure you have a clean prep. I'm sorry to say this, but I wouldn't trust any experiments done with a plasmid that might have some other random DNA in them. you wouldn't be likely to get a clean PCR product, cloning experiment, protein expression...pretty much any downstream app. could be considered contaminated.

like the boss always says 'the most expensive experiments are the ones you have to do twice'.
"it is a miracle that curiosity survives formal education" -A.E.

#7 GeorgeWolff

GeorgeWolff

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 294 posts
0
Neutral

Posted 03 March 2009 - 02:25 PM

aimikins is 100% right. Please consider the lack of technical rigor you're demonstrating for your PI.

#8 cotchy

cotchy

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 87 posts
1
Neutral

Posted 05 March 2009 - 07:36 AM

would any random DNa have shown up on a gel as i didnt see any extra bands compared to control prep from a while ago

i didnt use this plasmid and have started a new experiment but it would be interesting to know if gel showed up no other DNA but may still be some contamination?

#9 aimikins

aimikins

    Sunflower

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 117 posts
2
Neutral

Posted 05 March 2009 - 08:28 AM

yes, there could still be contamination. while agarose gels are very valuable tools, they are not the most sensitive no matter how well stained. I prefer to take a 'better safe than sorry' approach when it comes to DNA stocks.
"it is a miracle that curiosity survives formal education" -A.E.




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.